掌叶半夏有效提取物对宫颈癌细胞株的抑制作用及其机制研究
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摘要
宫颈癌是最常见的妇科恶性肿瘤之一,我国每年宫颈癌的新发病例数和死亡人数均居世界的前列。近年来随着人乳头状瘤病毒(human papilloma virus,简称HPV)感染率的升高,子宫颈癌的发病率呈明显上升趋势,而且高发年龄趋于年轻化,成为严重危害女性健康的疾病。
传统中药掌叶半夏(Pinellia pedatisecta schott)系天南星科半夏属掌叶半夏的块茎,产于我国大部分地区。上世纪七十年代,上海第一医学院妇产科医院(即现今的复旦大学附属妇产科医院)曾用掌叶半夏治疗子宫颈癌247例,初步证实了它的有效性、低毒性和安全性。但对该药的药理机制未进行过深入的研究。本课题拟在前期临床研究工作和现有文献的基础上,首次利用中科院上海药物所应用现代工艺提取的掌叶半夏有效提取物(Pinellia Extract,简称PE),观察其对体外培养的宫颈癌细胞株的作用,并研究可能的作用机制,希望为临床HPV感染以及宫颈癌癌前病变的辅助治疗寻求一种新的途径,最终预防宫颈癌的发生。
全文共分三个部分:1.PE对宮颈癌细胞株CaSki和HeLa的增殖抑制作用研究;2.PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用研究;3.PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53表汰的影响。
第一部分PE对宫颈癌细胞株CaSki和HeLa的增殖抑制作用研究
目的了解PE对宫颈癌细胞株CaSki和HeLa细胞增殖的影响,并研究可能的作用机制。
方法在宫颈癌细胞株CaSki和HeLa的培养基中加入PE,采用MTT比色法检测不同浓度PE作用不同时间对CaSki和HeLa细胞增殖的影响,同时观察PE对HeLa细胞对顺铂(DDP)敏感性的影响。采用倒置相差显微镜、扫描电镜和透射电镜观察CaSki和HeLa细胞经0.15mg/mL的PE作用后,细胞形态学和超微结构的变化。免疫细胞化学实验检测PE作用24小时后CaSki和HeLa细胞PCNA的表达变化;用In-cellWestern Blot方法检测不同浓度PE作用于CaSki和HeLa细胞15分钟后,磷酸化ERK的表达变化。
结果PE可抑制宫颈癌细胞株CaSki和HeLa的增殖,且抑制作用具有良好的时间和剂量依赖效应:同时发现低剂量PE可增强DDP对HeLa细胞的抑制作用。倒置相差显微镜下观察到PE作用后细胞出现皱缩并呈悬浮状态生长,进一步用扫描电镜和透射电镜观察发现PE作用后细胞表面的微绒毛明显减少,核固缩、胞浆空泡增多,出现凋亡表现。PE作用24小时后CaSki和HeLa细胞PCNA的表达明显下降;不同浓度PE作用于CaSki和HeLa细胞15分钟后,两种细胞磷酸化ERK的表达都出现不同程度的下降,并具有剂量依赖效应,即PE浓度越大,两种细胞磷酸化ERK的表达越低。
结论PE可以抑制人子宫颈癌细胞的增殖,对宫颈鳞癌和宫颈腺癌细胞株的抑制作用相似;同时PE可以提高HeLa细胞对顺铂的敏感性。PE抑制人子宫颈癌细胞增殖的作用机制包括抑制PCNA的表达、阻断MAPK/ERK信号转导通路。
第二部分PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用研究
目的了解PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用,并探讨可能的作用机制。
方法应用流式细胞仪检测PE作用不同时间后CaSki和HeLa的细胞凋亡和细胞周期的变化;用免疫荧光实验检测PE作用24小时后CaSki和HeLa细胞Bcl-2表达的变化。
结果流式细胞仪检测结果表明,1.15mg/mL的PE作用3天后CaSki和HeLa近半数细胞已经凋亡,作用5天后两种细胞的凋亡率高达70%以上,与对照组比较差异非常显著;而PE作用后两种细胞的细胞周期与对照组比较无明显区别。免疫荧光实验检测结果显示,0.15mg/mL PE作用24小时后,CaSki和HeLa细胞Bcl-2的表达均明显下降,与对照组比较差异显著。
结论PE可以促进宫颈癌细胞株CaSki和HeLa凋亡,且呈现时间依赖性;PE的促凋亡作用之一是通过降低Bcl-2的表达。
第三部分PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53的影响
目的了解PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53mRNA及蛋白表达的影响。
方法采用常规RT-PCR及Realtime RT-PCR检测PE作用24小时后宮颈癌细胞株CaSki和HeLa HPV E6及P53 mRNA的表达变化;采用Western blot检测PE作用24小时后宫颈癌细胞株CaSki和HeLa HPVE6及P53蛋白的表达变化。
结果常规RT-PCR及Realtime RT-PCR结果表明:0.1 5mg/mLPE作用于CaSki和HeLa细胞24小时后,P53mRNA表达水平明显增加,而HPV E6mRNA表达水平明显下降,与对照组比较差异非常显著;Western blot结果显示药物作用24小时后,两种细胞HPV E6蛋白的表达水平下降,与对照组比较差异显著,P53蛋白表达水平增加,但与对照组比较差异无统计学意义。
结论PE可以明显抑制宮颈癌细胞株CaSki和HeLa HPV E6mRNA和蛋白的表达;同时PE可以提高两种细胞P53 mRNA和蛋白的表达。这可能是PE抗宮颈癌的主要作用机制。
Cervical cancer is one of the most frequently gynecological neoplasma.With the prevalence of cervical HPV infection,the incidence of cervical cancer increased.More and more young women were suffered from cervical cancer.Cervical cancer is becoming one of severe diseases that could be harmful to women's health.
Pinellia pedatisecta schott is a traditional Chinese drug in mainly regions of our country.It has pungent flavor,warm in nature and poisonous.Possess effects of dispelling wind and relieving convulsion,drying dampness to eliminate phlegm,drying dampness to eliminate phlegm,eliminating stagnation.It can be used to cure thanatophidia bite,nameless swelling and toxicum.In 1970's,Pinellia pedatisecta schott had been used to treat 247 cases of cervical cancer in our hospital,the effective rate was 81.53%.The exact pharmacological mechanism by which Pinellia pedatisecta schott offered in this treatment had not been identified.In this study,we have investigated the inhibition effect of Pinellia extract(PE) on the growth of cervical cancer cells and the associated mechanisms.
There are three parts in the study:PartⅠEffects of PE on proliferation of the cervical cancer cell lines and mechanisms of action.PartⅡApoptosis induction Effect of PE on the cell lines of cervical cancer and the associated mechanisms.PartⅢEffects of PE on the expression of P53 and HPVE6 in the cell lines of cervical cancer.
PartⅠEffects of PE on proliferation of the cell lines of cervical cancer and mechanisms of action
Objective To observe the effects of PE on proliferation of cervical cancer cell lines CaSki and HeLa and its effect on the sensitivity of HeLa to DDP.Mechanisms of action were studied.
Methods Cervical cancer cell lines CaSki and HeLa were incubated with different concentrations of PE for different time.We tested the effects of PE on cell proliferation by MTT assay.The effects of PE on morphology and ultrastructure were studied by phase-contrast microscope and electron microscope.PCNA was determined by immunocytochemistry experiment.In-cell Westem blot was used to estimate the relative protein amounts of phosphorylated ERK of CaSki and HeLa cervical cancer cells treated with different concentrations of PE for 15 minutes.
Results PE could obviously inhibit the proliferation of CaSki and HeLa cells in a time and dose dependent manner.Low concentration of PE could elevate the inhibition effect of DDP on HeLa cells.Phase-contrast microscope and electron microscope revealed that the morphology and ultrastructure of CaSki and HeLa cells changed a lot.PCNA was down-regulated after treated with PE for 24 hours.PE could also inhibite phosphorylation of ERK in a dose dependent manner.
Conclusions PE could suppress proliferation of cervical cancer cells and induce morphology and ultrastructure changes of CaSki and HeLa.PE could also elevate the sensitivity of HeLa cells to DDP.Inhibition effect of PE on PCNA and blocking of MAPK/ERK signal transduction pathway were involed in the mechanisms.
PartⅡApoptosis induction effect of PE on the cell lines of cervical cancer and the associated mechanisms
Objective To investigate apopotosis induction effect of PE on the cell lines of cervical cancer and associated mechanisms.
Methods The apoptosis and cell cycle of cervical cancer ceils were detected with flowcytometry(FCM) and DAPI stain.Bcl-2 protein was detected with immunofluorescence experiment.
Results PE could induce cervical cancer cells apoptosis in a time dependent manner.The apoptosis rates of CaSki and HeLa cervical cancer cells were nearly 50%after treated with PE for 3 days,and up to more than 70%for 5days.But there was no change of cell cycle of cervical cancer cells after treated with PE.Apoptositic cells were also detected by DAPI. Bcl-2 protein was down-regulated after treated with PE for 24 hours.
Conclusions PE could induce cervical cancer cells apoptosis.Bcl-2 was involed in the apoptosis induction process.
PartⅢEffects of PE on the expression of P53 and HPV E6 in the cell lines of cervical cancer
Objective To study the effects of PE on the mRNA and protein expression of P53 and HPV E6 in cervical cancer cell lines CaSki and HeLa.
Methods RT-PCR and Realtime RT-PCR were used to estimate the relative amounts of P53 mRNA and HPV E6 mRNA.Western blot was used to measure P53 and HPV E6 protein levels before and after treatment with PE.
Results After treated with 0.15mg/ml PE for 24hs,the expression of P53 mRNA increased significantly.The expression of HPV E6 mRNA and protein decreased after PE's exposure significantly.The expression of P53 protein increased also,but there was no statistical significance compared with the control group.
Conclusion PE could up-regulate P53 gene's mRNA and protein expression,and could also down-regulate HPV E6 gene's mRNA and protein expression.The products of these genes could contribute to PE's effect on CaSki and HeLa,and then caused proliferation inhibition and apoptosis.
传统中药掌叶半夏(Pinellia pedatisecta schott)系天南星科半夏属掌叶半夏的块茎,产于我国大部分地区。上世纪七十年代,上海第一医学院妇产科医院(即现今的复旦大学附属妇产科医院)曾用掌叶半夏治疗子宫颈癌247例,初步证实了它的有效性、低毒性和安全性。但对该药的药理机制未进行过深入的研究。本课题拟在前期临床研究工作和现有文献的基础上,首次利用中科院上海药物所应用现代工艺提取的掌叶半夏有效提取物(Pinellia Extract,简称PE),观察其对体外培养的宫颈癌细胞株的作用,并研究可能的作用机制,希望为临床HPV感染以及宫颈癌癌前病变的辅助治疗寻求一种新的途径,最终预防宫颈癌的发生。
全文共分三个部分:1.PE对宮颈癌细胞株CaSki和HeLa的增殖抑制作用研究;2.PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用研究;3.PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53表汰的影响。
第一部分PE对宫颈癌细胞株CaSki和HeLa的增殖抑制作用研究
目的了解PE对宫颈癌细胞株CaSki和HeLa细胞增殖的影响,并研究可能的作用机制。
方法在宫颈癌细胞株CaSki和HeLa的培养基中加入PE,采用MTT比色法检测不同浓度PE作用不同时间对CaSki和HeLa细胞增殖的影响,同时观察PE对HeLa细胞对顺铂(DDP)敏感性的影响。采用倒置相差显微镜、扫描电镜和透射电镜观察CaSki和HeLa细胞经0.15mg/mL的PE作用后,细胞形态学和超微结构的变化。免疫细胞化学实验检测PE作用24小时后CaSki和HeLa细胞PCNA的表达变化;用In-cellWestern Blot方法检测不同浓度PE作用于CaSki和HeLa细胞15分钟后,磷酸化ERK的表达变化。
结果PE可抑制宫颈癌细胞株CaSki和HeLa的增殖,且抑制作用具有良好的时间和剂量依赖效应:同时发现低剂量PE可增强DDP对HeLa细胞的抑制作用。倒置相差显微镜下观察到PE作用后细胞出现皱缩并呈悬浮状态生长,进一步用扫描电镜和透射电镜观察发现PE作用后细胞表面的微绒毛明显减少,核固缩、胞浆空泡增多,出现凋亡表现。PE作用24小时后CaSki和HeLa细胞PCNA的表达明显下降;不同浓度PE作用于CaSki和HeLa细胞15分钟后,两种细胞磷酸化ERK的表达都出现不同程度的下降,并具有剂量依赖效应,即PE浓度越大,两种细胞磷酸化ERK的表达越低。
结论PE可以抑制人子宫颈癌细胞的增殖,对宫颈鳞癌和宫颈腺癌细胞株的抑制作用相似;同时PE可以提高HeLa细胞对顺铂的敏感性。PE抑制人子宫颈癌细胞增殖的作用机制包括抑制PCNA的表达、阻断MAPK/ERK信号转导通路。
第二部分PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用研究
目的了解PE对宫颈癌细胞株CaSki和HeLa的促凋亡作用,并探讨可能的作用机制。
方法应用流式细胞仪检测PE作用不同时间后CaSki和HeLa的细胞凋亡和细胞周期的变化;用免疫荧光实验检测PE作用24小时后CaSki和HeLa细胞Bcl-2表达的变化。
结果流式细胞仪检测结果表明,1.15mg/mL的PE作用3天后CaSki和HeLa近半数细胞已经凋亡,作用5天后两种细胞的凋亡率高达70%以上,与对照组比较差异非常显著;而PE作用后两种细胞的细胞周期与对照组比较无明显区别。免疫荧光实验检测结果显示,0.15mg/mL PE作用24小时后,CaSki和HeLa细胞Bcl-2的表达均明显下降,与对照组比较差异显著。
结论PE可以促进宫颈癌细胞株CaSki和HeLa凋亡,且呈现时间依赖性;PE的促凋亡作用之一是通过降低Bcl-2的表达。
第三部分PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53的影响
目的了解PE对宫颈癌细胞株CaSki和HeLa HPV E6及P53mRNA及蛋白表达的影响。
方法采用常规RT-PCR及Realtime RT-PCR检测PE作用24小时后宮颈癌细胞株CaSki和HeLa HPV E6及P53 mRNA的表达变化;采用Western blot检测PE作用24小时后宫颈癌细胞株CaSki和HeLa HPVE6及P53蛋白的表达变化。
结果常规RT-PCR及Realtime RT-PCR结果表明:0.1 5mg/mLPE作用于CaSki和HeLa细胞24小时后,P53mRNA表达水平明显增加,而HPV E6mRNA表达水平明显下降,与对照组比较差异非常显著;Western blot结果显示药物作用24小时后,两种细胞HPV E6蛋白的表达水平下降,与对照组比较差异显著,P53蛋白表达水平增加,但与对照组比较差异无统计学意义。
结论PE可以明显抑制宮颈癌细胞株CaSki和HeLa HPV E6mRNA和蛋白的表达;同时PE可以提高两种细胞P53 mRNA和蛋白的表达。这可能是PE抗宮颈癌的主要作用机制。
Cervical cancer is one of the most frequently gynecological neoplasma.With the prevalence of cervical HPV infection,the incidence of cervical cancer increased.More and more young women were suffered from cervical cancer.Cervical cancer is becoming one of severe diseases that could be harmful to women's health.
Pinellia pedatisecta schott is a traditional Chinese drug in mainly regions of our country.It has pungent flavor,warm in nature and poisonous.Possess effects of dispelling wind and relieving convulsion,drying dampness to eliminate phlegm,drying dampness to eliminate phlegm,eliminating stagnation.It can be used to cure thanatophidia bite,nameless swelling and toxicum.In 1970's,Pinellia pedatisecta schott had been used to treat 247 cases of cervical cancer in our hospital,the effective rate was 81.53%.The exact pharmacological mechanism by which Pinellia pedatisecta schott offered in this treatment had not been identified.In this study,we have investigated the inhibition effect of Pinellia extract(PE) on the growth of cervical cancer cells and the associated mechanisms.
There are three parts in the study:PartⅠEffects of PE on proliferation of the cervical cancer cell lines and mechanisms of action.PartⅡApoptosis induction Effect of PE on the cell lines of cervical cancer and the associated mechanisms.PartⅢEffects of PE on the expression of P53 and HPVE6 in the cell lines of cervical cancer.
PartⅠEffects of PE on proliferation of the cell lines of cervical cancer and mechanisms of action
Objective To observe the effects of PE on proliferation of cervical cancer cell lines CaSki and HeLa and its effect on the sensitivity of HeLa to DDP.Mechanisms of action were studied.
Methods Cervical cancer cell lines CaSki and HeLa were incubated with different concentrations of PE for different time.We tested the effects of PE on cell proliferation by MTT assay.The effects of PE on morphology and ultrastructure were studied by phase-contrast microscope and electron microscope.PCNA was determined by immunocytochemistry experiment.In-cell Westem blot was used to estimate the relative protein amounts of phosphorylated ERK of CaSki and HeLa cervical cancer cells treated with different concentrations of PE for 15 minutes.
Results PE could obviously inhibit the proliferation of CaSki and HeLa cells in a time and dose dependent manner.Low concentration of PE could elevate the inhibition effect of DDP on HeLa cells.Phase-contrast microscope and electron microscope revealed that the morphology and ultrastructure of CaSki and HeLa cells changed a lot.PCNA was down-regulated after treated with PE for 24 hours.PE could also inhibite phosphorylation of ERK in a dose dependent manner.
Conclusions PE could suppress proliferation of cervical cancer cells and induce morphology and ultrastructure changes of CaSki and HeLa.PE could also elevate the sensitivity of HeLa cells to DDP.Inhibition effect of PE on PCNA and blocking of MAPK/ERK signal transduction pathway were involed in the mechanisms.
PartⅡApoptosis induction effect of PE on the cell lines of cervical cancer and the associated mechanisms
Objective To investigate apopotosis induction effect of PE on the cell lines of cervical cancer and associated mechanisms.
Methods The apoptosis and cell cycle of cervical cancer ceils were detected with flowcytometry(FCM) and DAPI stain.Bcl-2 protein was detected with immunofluorescence experiment.
Results PE could induce cervical cancer cells apoptosis in a time dependent manner.The apoptosis rates of CaSki and HeLa cervical cancer cells were nearly 50%after treated with PE for 3 days,and up to more than 70%for 5days.But there was no change of cell cycle of cervical cancer cells after treated with PE.Apoptositic cells were also detected by DAPI. Bcl-2 protein was down-regulated after treated with PE for 24 hours.
Conclusions PE could induce cervical cancer cells apoptosis.Bcl-2 was involed in the apoptosis induction process.
PartⅢEffects of PE on the expression of P53 and HPV E6 in the cell lines of cervical cancer
Objective To study the effects of PE on the mRNA and protein expression of P53 and HPV E6 in cervical cancer cell lines CaSki and HeLa.
Methods RT-PCR and Realtime RT-PCR were used to estimate the relative amounts of P53 mRNA and HPV E6 mRNA.Western blot was used to measure P53 and HPV E6 protein levels before and after treatment with PE.
Results After treated with 0.15mg/ml PE for 24hs,the expression of P53 mRNA increased significantly.The expression of HPV E6 mRNA and protein decreased after PE's exposure significantly.The expression of P53 protein increased also,but there was no statistical significance compared with the control group.
Conclusion PE could up-regulate P53 gene's mRNA and protein expression,and could also down-regulate HPV E6 gene's mRNA and protein expression.The products of these genes could contribute to PE's effect on CaSki and HeLa,and then caused proliferation inhibition and apoptosis.
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17.Crean JK,Finlay D,Murphy M,et al.The role of p42/44 MAPK and protein kinase B in connective tissue growthfactor induced extracellular matrix protein production,cell migration,and actin cytoskeletal rearrangement in human mesangial cells[J].J Biol Chem,2002;277(46):44187-44194
18.Roberts RA,James NH,Cosulich SC.The role of proteinkinase B and mitogen- activated protein kinase in epidermal growth factor and tumor necrosis factor alpha-mediated rat hepatocyte survival and apoptosis[J].Hepatology,2000;31(2):420- 427
19.Gao R,Ball DK,Perbal B,et al.Connective tissue growth factor induces c-fos gene activation and cell proliferation through p44/42 MAP kinase in primary rat hepatic stellate cells[J].J Hepatol,2004;40(3):431-438
20.Lim JH,Lee ES,You HJ,et al.Ras-dependent induction of HIF-1 alpha785 via the Raf/MEK/ERK pathway:a novel mechanism of Ras-mediated tumor promotion[J].Oncogene,2004;23:9427-9431
21.卢建,余应年,徐仁宝。受体、信号转导系统与疾病。第1版 山东科学技术出版社,2001:249-254
22.Scott T.Eblen,J ill K.Slack,Michael J,et al.Rac-PAK signaling stimulates exracellular signal-regulated kinase(ERK) activation by regulating formation of MEK1-ERK complexes[J].Molecular and Cellular Biology,2002;22(17):6023-6033
23.Kuida K,Boucher DM.Functions of MAP kinases:insights from gene-targeting studies [J].J Biol Chem,2004;135(6):653-656
24.Daoud G,Amyot M,Rassart E,et al.ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta[J].J Physiol,2005;566(Pt 2):409-423
1.上海第一医学院妇产科医院,基础部化学教研组。掌叶半夏治疗子宫颈癌的研究。上海医学1978,1(1):13-16
2.李超荆,徐亚铭,孙月丽,归绥琪等。掌叶半夏抗子宫颈癌成分的研究[J]。上海第一医学院学报1981,8(6):421-423
3.石武法等。程序性细胞死亡。生命科学1995,2:20-22
4.PlasierB,et al.Automatic image anylysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay.J Immunol Method,1999;229:81-96
5.Koty PP,Zhang H,Franklin WA,et al.In vivo expression of p53 and bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.Lung Cancer,2002;35(2):155- 163
6.Patrick P,Koty,Haifan Zhang,et al.Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.Lung Cancer,1999;23:115-127
7.Kirch DG,Doseff A,Chau BN,et al.Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome-c.J Biol Chem,1999;274(30 ):21155-21161
8.Kiefer M C,Brauer M J,Powers V C,et al.Modulation of apoptosis by the widely distributed Bcl-2 homologue BAK.Nature,1995;374:736-739
9.Adams JM,Cory S.The Bcl-2 protein family:arbiters of cell survival.Science,1998;281(5381):1 322-1326
10.朱国萍,娄阳,徐冲。BCL-2蛋白家族与细胞凋亡。细胞生物学杂志2001,23(1):20-23
11.Reed JC,Miyashita T,Takayama S,et al.Bcl-2 family proteins:Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy.J Cell Biochem,1996;60:23-32
12.Kariya S,Ogawa Y,Yoshida S,et al.X-irradiation enhances the expression of Bcl-2 in HL-60 cells:the resulting effects on apoptosis and radiosensitivity.Int J Mol Med,1999;3:145-152
1.Steben M,Franco ED.Human papillomavirus infection:Epidemiology and pathophysiology.Gynecol Oncol,2007;(107):s2-s5
2.Bulk S,Berkhof J,Bulkmans NW,et al.Preferential risk of HPV16 for aquamous cell carcinoma and HPV18 for adenocarcinom of the cervix compared to women with normal cytology in the Netherlans. Br J Cancer,2006;94(1):171-175
3. Bernard HU. Gene expression of genital human papillomaviruses and considerations on potential antiviral app roaches.Antivir Ther, 2002;7: 219
4. Munger K, Baldwin A, Edwards KM, et al. Mechanisms of humanpap illomavirus induced oncogenesis. J Virol, 2004;78:11451
5. Parenti AR, Rugge M, Frizzera E, Ruol A, Noventa F, Ancona E, Ninfo V. P53 overexpression in the multistep process of esophageal carcinogenesis.Am J Surg Pathol, 1995;19:1418-1422
6. Bennett WP, Hollstein MC, Metcalf RA, Welsh JA, He A, Zhu SM, Kusters I, Resau JH, Trump BF, Lane DP. P53 mutation and protein accumulation during multistage human esophageal carcinogenesis. Cancer Res, 1992;52:6092-6097
7. Hopman AH, Smedts F, Dignef W, et al. Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalitis. J Pathol,2004;202(1):23-33
8. Melsheimer P, Vinokurova S, Wentzensen N, et al. DNA aneuploidy and integration of human papilomavirus type 16 E6/E7 oncogenes in intraepithelial meoplasia and invasive squamous cell carcinoma of cervix uteri. Clin Cancer Res,2004;10(9):3059-3063
9. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as an ubiquitin-protein ligase in the ubiquitination of P53. Cell, 1993 ;75:495-505
10. Gao Q, Srinivasan S, Boyer SN, et al. The E6 oncop roteins of high2risk human papillomavirus bind to a novel putative GAP protein, E6TP1 and target it for degradation.Mol CellBiol, 1999; 19(1):733-737
11. Duensing S, LeeL Y, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defectsand genomicin stability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci, 2000;97:10002
12. Jiang M, Milner J. Selective silencing of viral gene E6 and E7 expression in HPV-positive human cervical carcinoma cells using small interfering RNAs. Methods Mol Biol,2005;292:401-420
13. Cohen J.Public health,high hope and dilemmas for a cervical cancer vaccine.Science,2005;308(5722):618-621
14. Joura EA, Leodolter S, Hernandez A M, et al. Efficacy of a quadrivalent prophylactic human papillomavirus (type 6, 11, 16 and 18) L1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomized clinical trials. Lancet, 2007; 369(9574): 1693-1702
15. Gu W,Putral L,Hengst K, et al. Inhibition of cervical cancer cell growth in vitro and in vivi with lentiviral-vector delivered short hairpin RNA targeting hunman papillomavirus E6 and E7 oncogenes. Cancer Gene Ther,2006;13(11):1023-1032
16. Somasundaram KDS.Therapeutic potential of an adenovirus expressing p73beta, a p53 homologue, against human papiloma virus positive cervical cancer in vitro and in vivo.Cancer Biol Ther,2006;5(2):210-217
17. Perez-Cardenas E, Contreras-Paredes A, Cantu D, et al. The effects of DNA Methylation and histone deacetylase inhibitors on human papillomavirus early gege expression in cervical cancer, an in vitro and clinical study. Virol J, 2007; 26(4): 18-19
1.Graaf Y,Molijin A.Doornewaad H,et al.Human papilllomavirus and long-term risk of cervical neoplasia(J).Am J Epidemiol 2002,156(2):158-1641
2.Cain JM,Howett MK.Preventing cervical cancer.Science,2000,288:1753-1754
3.Munoz N.Human papillomavirus and cancer:the epidemiological evidence. Journal of Clinical Virology, 2000, 19:1-5
4. Hwang T S, Jeong J K, Park M, et al. Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray(J). Gyneol Oncol 2003,90(1):51 -56
5. Munoz N. Human papillomavirus and cancer: the epidemiological evidence. Journal of Clinical Virology, 2000, 19:1-5.
6. Dell G, Gaston K. Human papillomaviruses and their role in cervical cancer (J). Cell Mol Life Sci 2001,58(12-13);1923-1942
7. Altekruse SF, Lacey JV Jr, Brinton LA, et al. Comparison of human papilllomavirus genotypes,sexal,and reproductive risk factors of cervical adenocarcinoma and squamous cell carcinoma:Northeastern United States.Am J Obstet Gynecol 2003,188(3):657-663
8. Shyu JS, Chen CJ, Chiu CC, e t al.Correlation of human papillomavirus 16 and 18 with cervical neoplasia in histological typing and clinical stage in Taiwan: an in situ polymerase chian reaction approach. J Surg Oncol2001,78(2): 101-109
9. Schwartz SM, Daling JR, Shera KA, et al. Human papillomavirus and prognoasis of invasive cervical cancer: a population-based study. J Clin Oncol 2001,19(7): 1906-0915
10. Frazer IH. Prevention of cervical cancer through papillomavirus vaccination. Nat Rev Immunol, 2004; 4(1):46-54
11. Zur Hausen H.Papillomavirus and cancer: from basic studied to clinical application.Nat Rev Cancer, 200; 2(5):342-350
12. Wentzensen N, Vinokurova S, Doeberitz MK, et al. Systematic review of genomic integration sites of human papillomavirus geomes in epithelial dysplasia and invasive cancer of the female lower genital tract.Cancer Res,2004,64(11):3878-3884
13. Kornegay J R, Roger M, Davies P O, et al. International proficiency study of a consensus L1 PCR assay for the detection and typing of human papillomavirus DNA:evaluation of accuracy and intralaboratory and interboratory agreement(J). J Clin Microbiol 2003, 41(3):1080-1086
14. Pitts M, Clarke T. Human papillomavirus infections and risks of cervical cancer: what do women know (J). Health Educ Res 2002, 17(6):1080-1086
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16. Monis M, Ling M, Hung CF, et al. HPV DNA vaccines (J). Front Biosci 2003, 18:D55-D68
17. Cohen J. Public health, high hope and dilemmas for a cervical cancer vaccine.Science, 2005, 308(5722):618-621
18. Joura EA, Leodolter S, Hernandez AM, et al. Efficacy of a quadrivalent prophylactic human papillomavirus (type 6, 11, 16 and 18) L1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomized clinical trials. Lancet, 2007; 369(9574): 1693-1702
1.Parkin DM.Global cancer statistics in the year 2000[J].Lancet Oncol,2001,2(9):533
2.Howe SL,Vargas DE,Granada D,et al.Cervical cancer prevention in remote rural Nicaragua:Aprogram evalulation[J].Gyneol Oncol,2005,99(3):S232
3.Munoz N.Human papillomavirus and cancer:the epidemiological evidence[J].Journal of Clinical Virology,2000;19(1-2):1
4.Hwang TS,Jeong JK,Park M,et al.Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray[J].Gyneol Oncol,2003,90(1):51
5.Xu Q,Wang S,xi L,et al.Effects of human papillomavirus type 16 E7 protein on the growth of cervical carcinoma cells and immuno-escape through the TGF-β 1 signaling pathway[J].Gyneol Oncol,2006,101(1):132
6.上海第一医学院妇产科医院,基础部化学教研组.掌叶半夏治疗子宫颈癌的研究[J].上海医学,1978.1(1):13
7.上海第一医学院妇产科医院药剂科,基础部化学教研组.β-谷甾醇栓剂的研制[J].药学通 报,1980,15(2):23
8.李超荆,徐亚铭,孙月丽等.掌叶半夏抗子宫颈癌成分的研究[J].上海第一医学院学报,1981,8(6):421
9.朗景和.迎接子宫颈癌预防的全球挑战与机遇[J].中华妇产科杂志,2002,37(3):129
10.张颖,郎景和.人乳头瘤病毒疫苗在子宫颈病变预防和治疗中的应用[J].中华妇产科杂志,2006,4(3):214
11.孙光星,丁声颂,钱瑶君.掌叶半夏总蛋白的提取、化学分析和对小鼠S-180瘤株的抑制作用[J].上海医科大学学报,1992,19(1):17
12.朱铭伟,周抗美,丁声颂.掌叶半夏总蛋白对卵巢癌细胞株及人脐血造血细胞的作用[J].上海医科大学学报,1999,26(6):455
[1]Parkin DM.Global cancer statistics in the year 2000.Lancet Oncol,2001,2(9):533-43
[2]Munoz N.Human papillomavirus and cancer:the epidemiological evidence.Journal of Clinical Virology,2000,19:1-5
[3]Hwang TS,Jeong JK,Park M,et al.Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray.Gyneol Oncol,2003,90(1):51-56
[4]上海第一医学院妇产科医院,基础部化学教研组.掌叶半夏治疗子宫颈癌的研究.上海医学,1978,1(1):13-16
[5]上海第一医学院妇产科医院药剂科,基础部化学教研组.谷甾醇栓剂的研制.药学通报,1980,15(2):23-26
[6]李超荆,徐亚铭,孙月丽等.掌叶半夏抗子宫颈癌成分的研究.上海第一医学院学报,1981,8(6):421-423
[7]王滨,牛欣,韩旭华.中西医结合药理学研究方法探讨.中华中医药杂志2005,20(6):356-358
2.Munoz N.Human papillomavirus and cancer:the epidemiological evidence.Journal of Clinical Virology 2000,19:1-5
3.Hwang T S,Jeong J K,Park M,et al.Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray[J].Gyneol Oncol 2003,90(1):51-56
4.李晓静,李志宏,王玉芹等。掌叶半夏研究概况[J]。中医药信息,2004;21(1): 16-18
5.上海第一医学院妇产科医院,基础部化学教研组。掌叶半夏治疗子宫颈癌的研究[J]。上海医学,1978:1(1):13-16
6.李超荆,徐亚铭,孙月丽,归绥琪等。掌叶半夏抗子宫颈癌成分的研究[J]。上海第一医学院学报,1981;8(6):421-423
7.孙光星,丁声颂,钱瑶君等。掌叶半夏总蛋白的提取,化学分析和对小鼠S-180瘤株的抑制作用[J]。上海医科大学学报,1992;19(1):17-20
8.朱铭伟,周抗美,丁声颂等。掌叶半夏总蛋白对卵巢癌细胞株及人脐血造血细胞的作用[J]。上海医科大学学报,1999;26(6):455-457
9.黄必胜,陈科力。半夏类药材的不同提取物对体外培养的人肝癌细胞Be127402的生长抑制作用[J]。中草药,2007;20(7):834-836
10.朗景和。迎接子宫颈癌预防的全球挑战与机遇。中华妇产科杂志,2002;37(3):129-131
11.黄杰雄,黄致治。增殖细胞核抗原的研究进展。国外医学生理病理科学与临床分册,1994;14(1):9-11
12.Rochelle L,Garcia MD,Coltrera et al.Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed,embedded tissues[J].Am J pathols,1989;134(4):733-9
13.Takasaki Y,Fishwild D,Tan EM.Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera[J].J Exp Med,1984;159:98192
14.Kabayashi I,Matsuo K,Ishibashi Y,et al.Proliferative activity in dysplasia and carcinoma in situ of the uterine cervix analyzed by PCNA immunostaining and agNOR staining[J].Hum Pathol,1994;25:198-203
15.Viala E,Pouyssegar J.Regulation of tumor cell motility by ERK mitogen-activated protein kinases[J].Ann N Y Acad Sci,2004;1030:208-218
16.SU B,Karin M.Mitogen-activated protein kinase cascadesand regulation of gene expression[J].Curr Opinion Immunol,1996;8(3):402-411
17.Crean JK,Finlay D,Murphy M,et al.The role of p42/44 MAPK and protein kinase B in connective tissue growthfactor induced extracellular matrix protein production,cell migration,and actin cytoskeletal rearrangement in human mesangial cells[J].J Biol Chem,2002;277(46):44187-44194
18.Roberts RA,James NH,Cosulich SC.The role of proteinkinase B and mitogen- activated protein kinase in epidermal growth factor and tumor necrosis factor alpha-mediated rat hepatocyte survival and apoptosis[J].Hepatology,2000;31(2):420- 427
19.Gao R,Ball DK,Perbal B,et al.Connective tissue growth factor induces c-fos gene activation and cell proliferation through p44/42 MAP kinase in primary rat hepatic stellate cells[J].J Hepatol,2004;40(3):431-438
20.Lim JH,Lee ES,You HJ,et al.Ras-dependent induction of HIF-1 alpha785 via the Raf/MEK/ERK pathway:a novel mechanism of Ras-mediated tumor promotion[J].Oncogene,2004;23:9427-9431
21.卢建,余应年,徐仁宝。受体、信号转导系统与疾病。第1版 山东科学技术出版社,2001:249-254
22.Scott T.Eblen,J ill K.Slack,Michael J,et al.Rac-PAK signaling stimulates exracellular signal-regulated kinase(ERK) activation by regulating formation of MEK1-ERK complexes[J].Molecular and Cellular Biology,2002;22(17):6023-6033
23.Kuida K,Boucher DM.Functions of MAP kinases:insights from gene-targeting studies [J].J Biol Chem,2004;135(6):653-656
24.Daoud G,Amyot M,Rassart E,et al.ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta[J].J Physiol,2005;566(Pt 2):409-423
1.上海第一医学院妇产科医院,基础部化学教研组。掌叶半夏治疗子宫颈癌的研究。上海医学1978,1(1):13-16
2.李超荆,徐亚铭,孙月丽,归绥琪等。掌叶半夏抗子宫颈癌成分的研究[J]。上海第一医学院学报1981,8(6):421-423
3.石武法等。程序性细胞死亡。生命科学1995,2:20-22
4.PlasierB,et al.Automatic image anylysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay.J Immunol Method,1999;229:81-96
5.Koty PP,Zhang H,Franklin WA,et al.In vivo expression of p53 and bcl-2 and their role in programmed cell death in premalignant and malignant lung lesions.Lung Cancer,2002;35(2):155- 163
6.Patrick P,Koty,Haifan Zhang,et al.Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.Lung Cancer,1999;23:115-127
7.Kirch DG,Doseff A,Chau BN,et al.Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome-c.J Biol Chem,1999;274(30 ):21155-21161
8.Kiefer M C,Brauer M J,Powers V C,et al.Modulation of apoptosis by the widely distributed Bcl-2 homologue BAK.Nature,1995;374:736-739
9.Adams JM,Cory S.The Bcl-2 protein family:arbiters of cell survival.Science,1998;281(5381):1 322-1326
10.朱国萍,娄阳,徐冲。BCL-2蛋白家族与细胞凋亡。细胞生物学杂志2001,23(1):20-23
11.Reed JC,Miyashita T,Takayama S,et al.Bcl-2 family proteins:Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy.J Cell Biochem,1996;60:23-32
12.Kariya S,Ogawa Y,Yoshida S,et al.X-irradiation enhances the expression of Bcl-2 in HL-60 cells:the resulting effects on apoptosis and radiosensitivity.Int J Mol Med,1999;3:145-152
1.Steben M,Franco ED.Human papillomavirus infection:Epidemiology and pathophysiology.Gynecol Oncol,2007;(107):s2-s5
2.Bulk S,Berkhof J,Bulkmans NW,et al.Preferential risk of HPV16 for aquamous cell carcinoma and HPV18 for adenocarcinom of the cervix compared to women with normal cytology in the Netherlans. Br J Cancer,2006;94(1):171-175
3. Bernard HU. Gene expression of genital human papillomaviruses and considerations on potential antiviral app roaches.Antivir Ther, 2002;7: 219
4. Munger K, Baldwin A, Edwards KM, et al. Mechanisms of humanpap illomavirus induced oncogenesis. J Virol, 2004;78:11451
5. Parenti AR, Rugge M, Frizzera E, Ruol A, Noventa F, Ancona E, Ninfo V. P53 overexpression in the multistep process of esophageal carcinogenesis.Am J Surg Pathol, 1995;19:1418-1422
6. Bennett WP, Hollstein MC, Metcalf RA, Welsh JA, He A, Zhu SM, Kusters I, Resau JH, Trump BF, Lane DP. P53 mutation and protein accumulation during multistage human esophageal carcinogenesis. Cancer Res, 1992;52:6092-6097
7. Hopman AH, Smedts F, Dignef W, et al. Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalitis. J Pathol,2004;202(1):23-33
8. Melsheimer P, Vinokurova S, Wentzensen N, et al. DNA aneuploidy and integration of human papilomavirus type 16 E6/E7 oncogenes in intraepithelial meoplasia and invasive squamous cell carcinoma of cervix uteri. Clin Cancer Res,2004;10(9):3059-3063
9. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as an ubiquitin-protein ligase in the ubiquitination of P53. Cell, 1993 ;75:495-505
10. Gao Q, Srinivasan S, Boyer SN, et al. The E6 oncop roteins of high2risk human papillomavirus bind to a novel putative GAP protein, E6TP1 and target it for degradation.Mol CellBiol, 1999; 19(1):733-737
11. Duensing S, LeeL Y, Duensing A, et al. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defectsand genomicin stability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci, 2000;97:10002
12. Jiang M, Milner J. Selective silencing of viral gene E6 and E7 expression in HPV-positive human cervical carcinoma cells using small interfering RNAs. Methods Mol Biol,2005;292:401-420
13. Cohen J.Public health,high hope and dilemmas for a cervical cancer vaccine.Science,2005;308(5722):618-621
14. Joura EA, Leodolter S, Hernandez A M, et al. Efficacy of a quadrivalent prophylactic human papillomavirus (type 6, 11, 16 and 18) L1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomized clinical trials. Lancet, 2007; 369(9574): 1693-1702
15. Gu W,Putral L,Hengst K, et al. Inhibition of cervical cancer cell growth in vitro and in vivi with lentiviral-vector delivered short hairpin RNA targeting hunman papillomavirus E6 and E7 oncogenes. Cancer Gene Ther,2006;13(11):1023-1032
16. Somasundaram KDS.Therapeutic potential of an adenovirus expressing p73beta, a p53 homologue, against human papiloma virus positive cervical cancer in vitro and in vivo.Cancer Biol Ther,2006;5(2):210-217
17. Perez-Cardenas E, Contreras-Paredes A, Cantu D, et al. The effects of DNA Methylation and histone deacetylase inhibitors on human papillomavirus early gege expression in cervical cancer, an in vitro and clinical study. Virol J, 2007; 26(4): 18-19
1.Graaf Y,Molijin A.Doornewaad H,et al.Human papilllomavirus and long-term risk of cervical neoplasia(J).Am J Epidemiol 2002,156(2):158-1641
2.Cain JM,Howett MK.Preventing cervical cancer.Science,2000,288:1753-1754
3.Munoz N.Human papillomavirus and cancer:the epidemiological evidence. Journal of Clinical Virology, 2000, 19:1-5
4. Hwang T S, Jeong J K, Park M, et al. Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray(J). Gyneol Oncol 2003,90(1):51 -56
5. Munoz N. Human papillomavirus and cancer: the epidemiological evidence. Journal of Clinical Virology, 2000, 19:1-5.
6. Dell G, Gaston K. Human papillomaviruses and their role in cervical cancer (J). Cell Mol Life Sci 2001,58(12-13);1923-1942
7. Altekruse SF, Lacey JV Jr, Brinton LA, et al. Comparison of human papilllomavirus genotypes,sexal,and reproductive risk factors of cervical adenocarcinoma and squamous cell carcinoma:Northeastern United States.Am J Obstet Gynecol 2003,188(3):657-663
8. Shyu JS, Chen CJ, Chiu CC, e t al.Correlation of human papillomavirus 16 and 18 with cervical neoplasia in histological typing and clinical stage in Taiwan: an in situ polymerase chian reaction approach. J Surg Oncol2001,78(2): 101-109
9. Schwartz SM, Daling JR, Shera KA, et al. Human papillomavirus and prognoasis of invasive cervical cancer: a population-based study. J Clin Oncol 2001,19(7): 1906-0915
10. Frazer IH. Prevention of cervical cancer through papillomavirus vaccination. Nat Rev Immunol, 2004; 4(1):46-54
11. Zur Hausen H.Papillomavirus and cancer: from basic studied to clinical application.Nat Rev Cancer, 200; 2(5):342-350
12. Wentzensen N, Vinokurova S, Doeberitz MK, et al. Systematic review of genomic integration sites of human papillomavirus geomes in epithelial dysplasia and invasive cancer of the female lower genital tract.Cancer Res,2004,64(11):3878-3884
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